Regulation Of Apoptotic Cell Death Research Articles

Poration of mitochondrial membranes by amyloidogenic peptides and other biological toxins.

Mitochondria are essential organelles known to serve broad functions, including in cellular metabolism, calcium buffering, signaling pathways and the regulation of apoptotic cell death. Maintaining the integrity of the outer (OMM) and inner mitochondrial membranes (IMM) is vital for mitochondrial health. Cardiolipin (CL), a unique dimeric glycerophospholipid, is the signature lipid of energy-converting membranes. It plays a significant role in maintaining mitochondrial architecture and function, stabilizing protein complexes and facilitating efficient oxidative phosphorylation (OXPHOS) whilst regulating cytochrome c release from mitochondria. CL is especially enriched in the IMM and at sites of contact between the OMM and IMM. Disorders of protein misfolding, such as Alzheimer's and Parkinson's diseases, involve amyloidogenic peptides like amyloid-β, tau and α-synuclein, which form metastable toxic oligomeric species that interact with biological membranes. Electrophysiological studies have shown that these oligomers form ion-conducting nanopores in membranes mimicking the IMM's phospholipid composition. Poration of mitochondrial membranes disrupts the ionic balance, causing osmotic swelling, loss of the voltage potential across the IMM, release of pro-apoptogenic factors, and leads to cell death. The interaction between CL and amyloid oligomers appears to favour their membrane insertion and pore formation, directly implicating CL in amyloid toxicity. Additionally, pore formation in mitochondrial membranes is not limited to amyloid proteins and peptides; other biological peptides, as diverse as the pro-apoptotic Bcl-2 family members, gasdermin proteins, cobra venom cardiotoxins and bacterial pathogenic toxins, have all been described to punch holes in mitochondria, contributing to cell death processes. Collectively, these findings underscore the vulnerability of mitochondria and the involvement of CL in various pathogenic mechanisms, emphasizing the need for further research on targeting CL-amyloid interactions to mitigate mitochondrial dysfunction.

Fulvestrant alleviates cisplatin-induced acute kidney injury via repression of BNIP3-mediated apoptosis and autophagy.

Cisplatin-induced acute kidney injury (AKI) is a clinical disease characterized by a sudden loss of renal function within a few hours or days, due to cisplatin uptake. Fulvestrant is an oestrogen receptor alpha (ERα) antagonist used for endocrine therapy. However, the role of fulvestrant in cisplatin-induced AKI remains unclear. In this study, we investigated the effects of fulvestrant on the regulation of apoptotic cell death and autophagic response in cisplatin-induced AKI. The human kidney proximal tubule epithelial cell line (HK-2) was co-treated with fulvestrant and cisplatin. C57BL/6 mice were subcutaneously injected with fulvestrant and cisplatin was administered via intraperitoneal injection. First, cisplatin treatment increased ERα expression, apoptosis, and autophagy in HK-2 cells. Fulvestrant treatment decreased apoptosis and autophagy, which were accompanied by cisplatin treatment in HK-2 cells. Consistent with in vitro results, cisplatin treatment significantly increased ERα expression in vivo. Additionally, cisplatin treatment increased renal injury, apoptosis, and autophagy. Surprisingly, compared to that in the cisplatin-treated mice group, reduced cisplatin-induced renal injury, apoptosis, and autophagy was observed in the cisplatin+fulvestrant-treated mice group. In summary, these results suggest that fulvestrant plays an important role in cisplatin-induced AKI by decreasing apoptosis and autophagy.

Caspase 3-specific cleavage of MEK1 suppresses ERK signaling and sensitizes cells to stress-induced apoptosis.

Proper regulation of apoptotic cell death is crucial for normal development and homeostasis in multicellular organisms and is achieved by the balance between pro-apoptotic processes, such as caspase activation, and pro-survival signaling, such as extracellular signal-regulated kinase (ERK) activation. However, the functional interplay between these opposing signaling pathways remains incompletely understood. Here, we identified MAPK/ERK kinase (MEK) 1, a central component of the ERK pathway, as a specific substrate for the executioner caspase-3. During apoptosis, MEK1 is cleaved at an evolutionarily conserved Asp282 residue in the kinase domain, thereby losing its catalytic activity. Gene knockout experiments showed that MEK1 cleavage was mediated by caspase-3, but not by the other executioner caspases, caspase-6 or -7. Following exposure of cells to osmotic stress, elevated ERK activity gradually decreased, and this was accompanied by increased cleavage of MEK1. In contrast, the expression of a caspase-uncleavable MEK1(D282N) mutant in cells maintained stress-induced ERK activity and thereby attenuated apoptotic cell death. Thus, caspase-3-mediated, proteolytic inhibition of MEK1 sensitizes cells to apoptosis by suppressing pro-survival ERK signaling. Furthermore, we found that a RASopathy-associated MEK1(Y130C) mutation prevented this caspase-3-mediated proteolytic inactivation of MEK1 and efficiently protected cells from stress-induced apoptosis. Our data reveal the functional crosstalk between ERK-mediated cell survival and caspase-mediated cell death pathways and suggest that its dysregulation by a disease-associated MEK1 mutation is at least partly involved in the pathophysiology of congenital RASopathies.

Thiostrepton confers protection against reactive oxygen species-related apoptosis by restraining FOXM1-triggerred development of gastric cancer.

Gastric cancer is a leading cause of tumor-associated death worldwide. Metastasis and chemoresistance are crucial barriers for gastric cancer treatment. The Forkhead Box M1 (FOXM1) transcription factor has been reported as a promising treatment target for various types of tumors, but its effects on gastric cancer progression are not fully understood. In the present study, we found that FOXM1 expression levels were significantly up-regulated in human gastric cancer cell lines and tissues, and its expression was much higher in patients with metastasis. We then found that suppressing FOXM1 with its inhibitor thiostrepton (THIO) significantly reduced the proliferation of gastric cancer cells, while induced G0/G1 and apoptosis. Moreover, reactive oxygen species (ROS) production, mitochondrial impair and autophagy were remarkably provoked in gastric cancer cells treated with THIO, which were required for the regulation of apoptotic cell death. Furthermore, THIO exposure considerably suppressed the migration, invasion and angiogenesis in gastric cancer cells. The inhibitory effects of THIO on tumor growth and metastasis were confirmed in an established gastric cancer xenograft mouse model without detectable toxicity. Intriguingly, our in vitro studies showed that the anti-cancer effects of THIO on gastric cancer were almost abolished upon FOXM1 over-expression, indicating the necessity of FOXM1 suppression in THIO-inhibited tumor growth. In addition, higher FOXM1 expression was detected in gastric cancer cells with chemoresistance. Both in vitro and in vivo studies illustrated that THIO strongly promoted the drug-resistant gastric cancer cells to chemotherapies, proved by the considerably decreased cell proliferation and epithelial-mesenchymal transition (EMT) process. Together, these findings revealed that FOXM1 was a promising therapeutic target for gastric cancer treatment, and THIO exerted potential as an therapeutic agent for the disease.

Human gastric fibroblasts ameliorate A20-dependent cell survival in co-cultured gastric epithelial cells infected by Helicobacter pylori

Crosstalk within the gastric epithelium, which is closely in contact with stromal fibroblasts in the gastric mucosa, has a pivotal impact in proliferation, differentiation and transformation of the gastric epithelium. The human pathogen Helicobacter pylori colonises the gastric epithelium and represents a risk factor for gastric pathophysiology. Infection of H. pylori induces the activation of the transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), which is involved in the pro-inflammatory response but also in cell survival. In co-cultures with human gastric fibroblasts (HGF), we found that apoptotic cell death is reduced in the polarised human gastric cancer cell line NCI-N87 or in gastric mucosoids during H. pylori infection. Interestingly, suppression of apoptotic cell death in NCI-N87 cells involved an enhanced A20 expression regulated by NF-κB activity in response to H. pylori infection. Moreover, A20 acts as an important negative regulator of caspase-8 activity, which was suppressed in NCI-N87 cells during co-culture with gastric fibroblasts. Our results provide evidence for NF-κB-dependent regulation of apoptotic cell death in cellular crosstalk and highlight the protective role of gastric fibroblasts in gastric epithelial cell death during H. pylori infection.

Smart Design of Mitochondria-Targeted and ROS-Responsive CPI-613 Delivery Nanoplatform for Bioenergetic Pancreatic Cancer Therapy.

Mitochondria, as the powerhouse of most cells, are not only responsible for the generation of adenosine triphosphate (ATP) but also play a decisive role in the regulation of apoptotic cell death, especially of cancer cells. Safe potential delivery systems which can achieve organelle-targeted therapy are urgently required. In this study, for effective pancreatic cancer therapy, a novel mitochondria-targeted and ROS-triggered drug delivery nanoplatform was developed from the TPP-TK-CPI-613 (TTCI) prodrug, in which the ROS-cleave thioketal functions as a linker connecting mitochondrial targeting ligand TPP and anti-mitochondrial metabolism agent CPI-613. DSPE-PEG2000 was added as an assistant component to increase accumulation in the tumor via the EPR effect. This new nanoplatform showed effective mitochondrial targeting, ROS-cleaving capability, and robust therapeutic performances. With active mitochondrial targeting, the formulated nanoparticles (TTCI NPs) demonstrate much higher accumulation in mitochondria, facilitating the targeted delivery of CPI-613 to its acting site. The results of in vitro antitumor activity and cell apoptosis revealed that the IC50 values of TTCI NPs in three types of pancreatic cancer cells were around 20~30 µM, which was far lower than those of CPI-613 (200 µM); 50 µM TTCI NPs showed an increase in apoptosis of up to 97.3% in BxPC3 cells. Therefore, this mitochondria-targeted prodrug nanoparticle platform provides a potential strategy for developing safe, targeting and efficient drug delivery systems for pancreatic cancer therapy.

Novel Synthesized N-Ethyl-Piperazinyl-Amides of C2-Substituted Oleanonic and Ursonic Acids Exhibit Cytotoxic Effects through Apoptotic Cell Death Regulation.

A series of novel hybrid chalcone N-ethyl-piperazinyl amide derivatives of oleanonic and ursonic acids were synthesized, and their cytotoxic potential was evaluated in vitro against the NCI-60 cancer cell line panel. Compounds 4 and 6 exhibited the highest overall anticancer activity, with GI50 values in some cases reaching nanomolar values. Thus, the two compounds were further assessed in detail in order to identify a possible apoptosis- and antiangiogenic-based mechanism of action induced by the assessed compounds. DAPI staining revealed that both compounds induced nuclei condensation and overall cell morphological changes consistent with apoptotic cell death. rtPCR analysis showed that up-regulation of pro-apoptotic Bak gene combined with the down-regulation of the pro-survival Bcl-XL and Bcl-2 genes caused altered ratios between the pro-apoptotic and anti-apoptotic proteins’ levels, leading to overall induced apoptosis. Molecular docking analysis revealed that both compounds exhibited high scores for Bcl-XL inhibition, suggesting that compounds may induce apoptotic cell death through targeted anti-apoptotic protein inhibition, as well. Ex vivo determinations showed that both compounds did not significantly alter the angiogenesis process on the tested cell lines.

A mutation in desmin makes skeletal muscle less vulnerable to acute muscle damage after eccentric loading in rats.

Desminopathy is the most common intermediate filament disease in humans. The most frequent mutation causing desminopathy in patients is a R350P DES missense mutation. We have developed a rat model with an analogous mutation in R349P Des. To investigate the role of R349P Des in mechanical loading, we stimulated the sciatic nerve of wild‐type littermates (WT) (n = 6) and animals carrying the mutation (MUT) (n = 6) causing a lengthening contraction of the dorsi flexor muscles. MUT animals showed signs of ongoing regeneration at baseline as indicated by a higher number of central nuclei (genotype: P < .0001). While stimulation did not impact central nuclei, we found an increased number of IgG positive fibers (membrane damage indicator) after eccentric contractions with both genotypes (stimulation: P < .01). Interestingly, WT animals displayed a more pronounced increase in IgG positive fibers with stimulation compared to MUT (interaction: P < .05). In addition to altered histology, molecular signaling on the protein level differed between WT and MUT. The membrane repair protein dysferlin decreased with eccentric loading in WT but increased in MUT (interaction: P < .05). The autophagic substrate p62 was increased in both genotypes with loading (stimulation: P < .05) but tended to be more elevated in WT (interaction: P = .05). Caspase 3 levels, a central regulator of apoptotic cell death, was increased with stimulation in both genotypes (stimulation: P < .01) but more so in WT animals (interaction: P < .0001). Overall, our data indicate that R349P Des rats have a lower susceptibility to structural muscle damage of the cytoskeleton and sarcolemma with acute eccentric loading.

The role of mitochondria in metabolic disease: a special emphasis on heart dysfunction.

Metabolic diseases (MetDs) embrace a series of pathologies characterized by abnormal body glucose usage. The known diseases included in this group are metabolic syndrome, prediabetes and diabetes mellitus types 1 and 2. All of them are chronic pathologies that present metabolic disturbances and are classified as multi-organ diseases. Cardiomyopathy has been extensively described in diabetic patients without overt macrovascular complications. The heart is severely damaged during the progression of the disease; in fact, diabetic cardiomyopathies are the main cause of death in MetDs. Insulin resistance, hyperglycaemia and increased free fatty acid metabolism promote cardiac damage through mitochondria. These organelles supply most of the energy that the heart needs to beat and to control essential cellular functions, including Ca2+ signalling modulation, reactive oxygen species production and apoptotic cell death regulation. Several aspects of common mitochondrial functions have been described as being altered in diabetic cardiomyopathies, including impaired energy metabolism, compromised mitochondrial dynamics, deficiencies in Ca2+ handling, increases in reactive oxygen species production, and a higher probability of mitochondrial permeability transition pore opening. Therefore, the mitochondrial role in MetD-mediated heart dysfunction has been studied extensively to identify potential therapeutic targets for improving cardiac performance. Herein we review the cardiac pathology in metabolic syndrome, prediabetes and diabetes mellitus, focusing on the role of mitochondrial dysfunctions.

Contribution of Yeast Studies to the Understanding of BCL-2 Family Intracellular Trafficking.

BCL-2 family members are major regulators of apoptotic cell death in mammals. They form an intricate regulatory network that ultimately regulates the release of apoptogenic factors from mitochondria to the cytosol. The ectopic expression of mammalian BCL-2 family members in the yeast Saccharomyces cerevisiae, which lacks BCL-2 homologs, has been long established as a useful addition to the available models to study their function and regulation. In yeast, individual proteins can be studied independently from the whole interaction network, thus providing insight into the molecular mechanisms underlying their function in a living context. Furthermore, one can take advantage of the powerful tools available in yeast to probe intracellular trafficking processes such as mitochondrial sorting and interactions/exchanges between mitochondria and other compartments, such as the endoplasmic reticulum that are largely conserved between yeast and mammals. Yeast molecular genetics thus allows the investigation of the role of these processes on the dynamic equilibrium of BCL-2 family members between mitochondria and extramitochondrial compartments. Here we propose a model of dynamic regulation of BCL-2 family member localization, based on available evidence from ectopic expression in yeast.

Impact of Fas/Fasl Gene Polymorphisms on Susceptibility Risk and Imatinib Mesylate Treatment Response in Chronic Myeloid Leukaemia Patients

Background:The FAS mediated apoptosis pathway involving the FAS and FASL genes plays a crucial role in the regulation of apoptotic cell death and imatinib mesylate (IM) mechanism of action. Promoter polymorphisms FAS-670 A>G and FAS-844 T>C which alter the transcriptional activity of these genes may grant a risk to develop cancer and revamp the drug activities towards the cancer cell. We investigated the association of these two polymorphisms with the susceptibility risk and IM treatment response in Malaysian chronic myeloid leukaemia (CML) patients. Methods:This is a retrospective study, which included 93 CML patients and 98 controls. The polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) method was used to genotype the FAS and FASL polymorphisms. Data nanlysis was done using SPSS Version 22. The associations of the genotypes with susceptibility risk and IM response in CML patients were assessed by means of logistic regression analysis and deriving odds ratio with 95% CI. Results:We observed a significant association between FASL-844T>C polymorphism and CML susceptibility risk and IM response. Variant C allele and FASL-844 CC variant genotype carriers had significantly higher risk for CML susceptibility (OR 1.756, CI 1.163-2.652, p=0.007 and OR 2.261, CI 1.013-5.047, p=0.047 respectively). Conversely, the heterozygous genotype FASL-844 TC conferred lower risk for CML susceptibility (OR 0.379, CI 0.176-0.816, p=0.013). The heterozygous and homozygous variant genotypes and variant C alleles were found to confer a lower risk for the development of IM resistance with OR 0.129 (95% CI: 0.034-0.489 p=0.003), OR 0.257 (95% CI: 0.081-0.818, p=0.021), and OR 0.486 (95% CI: 0.262-0.899, p=0.021) respectively. We also found that FAS-670 A>G polymorphism was not associated with CML susceptibility risk or IM response. Conclusion:The genetic polymorphism FASL-844 T>C may contribute to the CML susceptibility risk and also IM treatment response in CML patients. Accodringly, it may be useful as a biomarker for predicting CML susceptibility risk and IM resistance.

Caspases from scleractinian coral show unique regulatory features

Coral reefs are experiencing precipitous declines around the globe with coral diseases and temperature-induced bleaching being primary drivers of these declines. Regulation of apoptotic cell death is an important component in the coral stress response. Although cnidaria are known to contain complex apoptotic signaling pathways, similar to those in vertebrates, the mechanisms leading to cell death are largely unexplored. We identified and characterized two caspases each from Orbicella faveolata, a disease-sensitive reef-building coral, and Porites astreoides, a disease-resistant reef-building coral. The caspases are predicted homologs of the human executioner caspases-3 and -7, but OfCasp3a (Orbicella faveolata caspase-3a) and PaCasp7a (Porites astreoides caspase-7a), which we show to be DXXDases, contain an N-terminal caspase activation/recruitment domain (CARD) similar to human initiator/inflammatory caspases. OfCasp3b (Orbicella faveolata caspase-3b) and PaCasp3 (Porites astreoides caspase-3), which we show to be VXXDases, have short pro-domains, like human executioner caspases. Our biochemical analyses suggest a mechanism in coral which differs from that of humans, where the CARD-containing DXXDase is activated on death platforms but the protease does not directly activate the VXXDase. The first X-ray crystal structure of a coral caspase, of PaCasp7a determined at 1.57 Å resolution, reveals a conserved fold and an N-terminal peptide bound near the active site that may serve as a regulatory exosite. The binding pocket has been observed in initiator caspases of other species. These results suggest mechanisms for the evolution of substrate selection while maintaining common activation mechanisms of CARD-mediated dimerization.

HDAC3-ERα Selectively Regulates TNF-α-Induced Apoptotic Cell Death in MCF-7 Human Breast Cancer Cells via the p53 Signaling Pathway.

Tumor necrosis factor-α (TNF-α) plays a significant role in inflammation and cancer-related apoptosis. We identified a TNF-α-mediated epigenetic mechanism of apoptotic cell death regulation in estrogen receptor-α (ERα)-positive human breast cancer cells. To assess the apoptotic effect of TNF-α, annexin V/ propidium iodide (PI) double staining, cell viability assays, and Western blotting were performed. To elucidate this mechanism, histone deacetylase (HDAC) activity assay and immunoprecipitation (IP) were conducted; the mechanism was subsequently confirmed through chromatin IP (ChIP) assays. Finally, we assessed HDAC3–ERα-mediated apoptotic cell death after TNF-α treatment in ERα-positive human breast cancer (MCF-7) cells via the transcriptional activation of p53 target genes using luciferase assay and quantitative reverse transcription PCR. The TNF-α-induced selective apoptosis in MCF-7 cells was negatively regulated by the HDAC3–ERα complex in a caspase-7-dependent manner. HDAC3 possessed a p53-binding element, thus suppressing the transcriptional activity of its target genes. In contrast, MCF-7 cell treatment with TNF-α led to dissociation of the HDAC3–ERα complex and substitution of the occupancy on the promoter by the p53–p300 complex, thus accelerating p53 target gene expression. In this process, p53 stabilization was accompanied by its acetylation. This study showed that p53-mediated apoptosis in ERα-positive human breast cancer cells was negatively regulated by HDAC3–ERα in a caspase-7-dependent manner. Therefore, these proteins have potential application in therapeutic strategies.

Apoptotic cell death regulation in neurons.

Apoptosis plays a major role in shaping the developing nervous system during embryogenesis as neuronal precursors differentiate to become post-mitotic neurons. However, once neurons are incorporated into functional circuits and become mature, they greatly restrict their capacity to die via apoptosis, thus allowing the mature nervous system to persist in a healthy and functional state throughout life. This robust restriction of the apoptotic pathway during neuronal differentiation and maturation is defined by multiple unique mechanisms that function to more precisely control and restrict the intrinsic apoptotic pathway. However, while these mechanisms are necessary for neuronal survival, mature neurons are still capable of activating the apoptotic pathway in certain pathological contexts. In this review, we highlight key mechanisms governing the survival of post-mitotic neurons, while also detailing the physiological and pathological contexts in which neurons are capable of overcoming this high apoptotic threshold.

Association of caspase 8 promoter variants and haplotypes with the risk of breast cancer and its molecular profile in an Iranian population: A case‐control study

Caspase 8 (CASP8) gene plays a key role in the regulationof apoptotic cell death. Expression variation in this gene has been associated with the risk of breast cancer. The aim of this study was to investigate the association of rs3834129 and rs3769821, as functional variants, and their haplotypes with molecular profile as well as the risk of breast cancer in an Iranian population. A case-control study was conducted on 812 participants including 293 breast cancer patients and 519 healthy controls. Genotyping was performed by polymerase chain reaction-based methods. Statistical analysis was performed using SPSS Ver16. The association between polymorphisms and haplotypes with the risk of breast cancer was estimated by calculating odds ratios (OR) and chi-square (χ2 ) tests. In comparison with ins allele (I) of rs3834129, carriers of del allele (D) showed a lower risk of breast cancer (OR, 0.65; 95% confidence interval [CI], 0.49-0.87; P = 0.004). The multivariate logistic regression model indicated DD genotype as an independent factor for a decreased risk of breast cancer in our population (OR, 0.18; 95% CI, 0.06-0.58; P = 0.004). Also, the C allele of rs3769821 was associated with a 43% increased risk of breast cancer (P = 0.005); however, after adjustment for confounding factors, no association with rs3769821 and breast cancer was observed. In addition, D-T haplotype and diplotype presented protective effects (P < 0.05). Our results indicate that genetic variations in the promoter region of CASP8 gene, especially rs3834129, may serve as a genetic risk factor for breast cancer in an Iranian population.

Carboxylated carbon nanomaterials in cell cycle and apoptotic cell death regulation

Carbon nanomaterials, include carbon nanotubes and graphene nanosheets, have drawn an increasing amount of attention because of their potential applications in daily life or in providing novel therapeutic possibilities for treating diseases. However, the overall biocompatibility, the potential toxic effects of carbon nanomaterials toward human cells, and their modulations in cellular mechanism, are not fully understood. Herein, four types of carbon nanomaterials, include long and short carbon nanotubes and graphene nanosheets, at low and high concentrations, were functionalized and dispersed in the biocompatible buffer for assessment. The surface structure, the morphology, and chemical composition of carbon nanomaterials were characterized. Also, biological assays investigating cellular viability, vitality, cell cycle, and apoptotic cell death were applied on cells co-incubated with nanomaterials, to evaluate the biocompatibility of these nanomaterials in human cells. Our data suggested that even though co-incubation of nanomaterials did not seem to affect the viability of cells notably, high concentrations (50 ug/ml) of SW could lead to unhealthy cells, and we observed dramatic G2 arrest effect mediated by p21 induction in high SW incubated cells. Other nanomaterials at high concentration may also alter cell cycle profile of the cells. In summary, our data demonstrated that these nanomaterials could regulate cell cycle and lead to apoptosis at high concentrations, and the underling molecular mechanisms have been addressed. Caution should be taken on their concentration when nanomaterials are in used in future medical applications.

Homology Modeling and Docking Studies of Bcl-2 and Bcl-xL with Small Molecule Inhibitors: Identification and Functional Studies.

Apoptosis is a vital physiological process, which is observed in various biological events. The anti-apoptotic and pro-apoptotic members of Bcl-2 family are the most characterized proteins which are involved in the regulation of apoptotic cell death. The anti-apoptotic proteins such as Bcl-2 and Bcl-xL prevent apoptosis, whereas pro-apoptotic members like Bax and Bak, elicit the release of caspases from death antagonists inducing apoptosis. Thus, the Bcl-2 family of proteins play a vital role in controlling programmed cell death. Over expression of anti-apoptotic Bcl-2 proteins are often directly associated with various kinds of cancer. Developing suitable inhibitors for controlling the elevated levels of these proteins got much attention in last decade. Structural biology techniques such as Nuclear Magnetic Resonance (NMR) spectroscopy, X-ray crystallography, homology modeling and molecular docking play a significant role in identifying the key inhibitors of these proteins. The authors have developed and tested successfully, several series of indole pharmacore containing inhibitors for Bcl-2 and Bcl-xL proteins based on the homology modeling, docking and suitable biochemical and apoptosis assays. This review provides a summary of potential inhibitor molecules developed for Bcl-2 and Bcl-xL proteins, as well as the the key residues of these proteins interacting with potential drug molecules. The present appraisal also focuses on the role of computational algorithms in developing potential drug molecules,with more emphasis on the role of homology modeling and docking studies in developing inhibitors for Bcl- 2, and Bcl-xL proteins in cancer therapy.

Mitochondrial chaotic dynamics: Redox-energetic behavior at the edge of stability

Mitochondria serve multiple key cellular functions, including energy generation, redox balance, and regulation of apoptotic cell death, thus making a major impact on healthy and diseased states. Increasingly recognized is that biological network stability/instability can play critical roles in determining health and disease. We report for the first-time mitochondrial chaotic dynamics, characterizing the conditions leading from stability to chaos in this organelle. Using an experimentally validated computational model of mitochondrial function, we show that complex oscillatory dynamics in key metabolic variables, arising at the “edge” between fully functional and pathological behavior, sets the stage for chaos. Under these conditions, a mild, regular sinusoidal redox forcing perturbation triggers chaotic dynamics with main signature traits such as sensitivity to initial conditions, positive Lyapunov exponents, and strange attractors. At the “edge” mitochondrial chaos is exquisitely sensitive to the antioxidant capacity of matrix Mn superoxide dismutase as well as to the amplitude and frequency of the redox perturbation. These results have potential implications both for mitochondrial signaling determining health maintenance, and pathological transformation, including abnormal cardiac rhythms.

Abstract WMP38: Acute Administration of Chlorpromazine and Promethazine Reduces Brain Apoptosis in Ischemic Stroke

Introduction: In recent studies conducted by our lab, acute administration of chlorpromazine and promethazine (C+P) in ischemic stroke has been shown, through the induction of a metabolically-slowed, hibernation-like state, to reduce infarct size and neurological deficit, and thus to exert a neuroprotective effect on infracted subjects. Although its mechanism has not been fully elucidated, this effect is associated with alterations in the expression of protein kinase C-δ (PKC-δ) and Akt. Because PKC-δ and Akt are also known to be involved in the regulation of apoptotic cell death after ischemic stroke, we hypothesized that treatment with C+P would result in reductions in post-ischemic apoptosis. Additionally, because the neuroprotective effect of C+P is associated with reductions in NADPH oxidase (NOX) expression and activity, and because of the involvement of NOX in the PKC-Akt apoptotic pathway, we hypothesized that adding a NOX inhibitor would potentiate the anti-apoptotic effect of C+P. Methods: 8mg/kg C+P with or without a NOX inhibitor was administered intraperitoneally to Sprague-Dawley rats subjected to 2h MCAO, followed by 6 or 24h reperfusion. The amount of apoptotic cell death in control, non-treatment ischemic, and all treatment ischemic groups was quantified using ELISA for cytoplasmic histone-associated DNA fragments. Levels of pro-apoptotic (caspase-3, BAX, and AIF) and anti-apoptotic (Bcl-2 and Bcl-xL) proteins were determined by Western blot. Results: Rats treated with C+P followed by 6h and 24h reperfusion demonstrated significant reductions in apoptotic cell death, increased expression of anti-apoptotic proteins, and decreased expression of pro-apoptotic proteins, compared with counterparts in the non-treatment ischemic group. Rats treated with C+P together with a NOX inhibitor exhibited further reductions in apoptotic cell death. Conclusion: These results demonstrate that C+P administration causes significant reductions in apoptotic cell death following ischemic stroke, when measured both at 6 and 24h reperfusion, and that addition of a NOX inhibitor potentiates this reduction. Therefore, the neuroprotective function observed upon C+P administration appears to involve the prevention of post-stroke apoptosis.

Impact of cell death pathway genes Fas 21377AA and FasL 2844CC polymorphisms on the risk of developing non-small cell lung cancer

BackgroundThe signalling pathway Fas and FasL system plays a fundamental role in the regulation of apoptotic cell death and any disturbance of this pathway has been shown to promote immune escape and tumorigenesis. Many types of cancers show reduced expression of FAS and elevated FasL expression. The Fas21377G/A, and FasL2844T/C polymorphisms might be associated with increased risk of lung cancer. ObjectiveThe interplay between genetic polymorphisms could participate in cancer development. This study aimed to examine the contribution of Fas21377AA and FasL2844CC genotypes to risk of developing lung cancer. Subjects and methodsA case-control study was conducted on 20 non small cell lung cancer (NSCLC) cases and 40 controls. Genotyping of Fas 21377AA and FasL 2844CC Single nucleotide polymorphisms (SNPs) by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) were done to all subjects. ResultsThe distribution of Fas and FasL genotypes showed a higher frequency of Fas AA genotype among patients compared to controls with an increased risk of lung cancer (OR 5.28; CI:1.35–20.65, P value .01). No statistically significant difference was found between patients and controls groups in respect to FasL genotypes. Gene to gene interaction between Fas and FasL genotypes showed significant differences between the patients and controls groups. As regards the combination between FasL TT+CT & Fas AA, FasL CC & Fas GG+GA and FasL CC & Fas AA genotypes where patients carrying FasL CC or Fas AA genotypes have increased risk to develop lung cancer, (OR 10.28, 95% CI; 1.68–62.74, P value .01), (OR 72, 95% CI; 5.55–132.99, P value .001) and (OR 9, 95% CI; 1.5–53.86, P value .01) respectively. The FasL-CC genotype showed 2.25 folds increased risk to develop lung cancer in non-smoker patients, P = .008. No correlation was found between the pathological types, the stage of lung cancer and the Fas and FasL genotypes. ConclusionThe interaction of the cell death pathway genes Fas and FasL polymorphisms could be associated with the risk of lung cancer, in the same respect Fas AA genotype could also potentiate this risk.